In recent years, mass spectrometry has gained popularity as a tool for identifying microorganisms due to its increased accuracy and shortened time-to-result when compared to traditional methods for identifying microorganisms. To date, the most common mass spectrometry method used for microbial identification is matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In MALDI-TOF, cells of an unknown microorganism are mixed with a suitable ultraviolet light absorbing matrix solution and are allowed to dry on a sample plate. Alternatively, an extract of microbial cells is used instead of the intact cells. After transfer to the ion source of a mass spectrometer, a laser beam is directed to the sample for desorption and ionization of the proteins and time-dependent mass spectral data is collected.
The mass spectrum of a microorganism produced by MALDI-TOF methods reveals a number of peaks from intact peptides, proteins, protein fragments, and other molecules that constitute the microorganism's “fingerprint”. This method relies on the pattern matching of the peak profiles in the mass spectrum of an unknown microorganism to a reference database comprising a collection of mass spectra for known microorganisms obtained using essentially the same experimental conditions. The better the match between the spectrum of the isolated microorganism and a spectrum in the reference database, the higher the confidence level in identification of the organism at the genus, species, or in some cases, subspecies level. Because the method relies upon matching the patterns of peaks in MALDI-TOF mass spectra, there is no requirement to identify or otherwise characterize the proteins represented in the spectrum of the unknown microorganism in order to identify it.
Although MALDI-TOF methods are rapid and cost effective, they have limitations that restrict the range of applications to pathogen characterization and identification including but not limited to virulence detection and quantitation, resistance marker determination, strain matching, and antibiotic susceptibility testing to name a few. The information content within a MALDI mass spectrum reflects the most abundant and ionizable proteins which are generally limited to ribosomal proteins at the experimental conditions used. Because ribosomal proteins are highly conserved among prokaryotes, differentiation of closely related microorganisms by MALDI-TOF is limited. In this case many of the ribosomal proteins across closely related species contain either the same or slightly different amino acid sequences (i.e. single amino acid substitutions) that cannot be effectively differentiated with low resolution mass spectrometers. Moreover, determination of strain and/or serovar type, antibiotic resistance, antibiotic susceptibility, virulence or other important characteristics relies upon the detection of protein markers other than ribosomal proteins which further limits the application of MALDI-TOF for microbial analysis. Laboratories using MALDI-TOF for identification of microorganisms must use other methods to further characterize the identified microbes. In addition, the MALDI-TOF method's reliance upon matching spectral patterns requires a pure culture for high quality results and thus is not generally suitable for direct testing, mixed cultures, blood culture, or other complex samples containing different microorganisms.
Several other mass spectrometry methods for detection of microorganisms have been used. For example, mass spectrometry-based protein sequencing methods have been described wherein liquid chromatography is coupled to tandem mass spectrometry (LC-MS/MS) and sequence information is obtained from enzymatic digests of proteins derived from the microbial sample. This approach, termed “bottom-up” proteomics, is a widely practiced method for protein identification. The method can provide identification to the subspecies or strain level as chromatographic separation allows the detection of additional proteins other than just ribosomal proteins, including those useful for characterization of antibiotic resistance markers and virulence factors.
In contrast to “bottom-up” proteomics, “top-down” proteomics refers to methods of analysis in which protein samples are introduced intact into a mass spectrometer, without enzymatic, chemical or other means of digestion. Top-down analysis enables the study of the intact protein, allowing identification, primary structure determination and localization of post-translational modifications (PTMs) directly at the protein level. Top-down proteomic analysis typically consists of introducing an intact protein into the ionization source of a mass spectrometer, fragmenting the protein ions and measuring the mass-to-charge ratios and abundances of the various fragments so-generated. The resulting fragmentation is many times more complex than a peptide fragmentation, which may, in the absence of the methods taught herein, necessitate the use of a mass spectrometer with very high mass accuracy and resolution capability in order to interpret the fragmentation pattern with acceptable certainty. The interpretation generally includes comparing the observed fragmentation pattern to either a protein sequence database that includes compiled experimental fragmentation results generated from known samples or, alternatively, to theoretically predicted fragmentation patterns. For example, Liu et al. (“Top-Down Protein Identification/Characterization of a Priori Unknown Proteins via Ion Trap Collision-Induced Dissociation and Ion/Ion Reactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer”, Anal. Chem. 2009, 81, 1433-1441) have described top-down protein identification and characterization of both modified and unmodified unknown proteins with masses up to ≈28 kDa.
An advantage of a top-down analysis over a bottom-up analysis is that a protein may be identified directly, rather than inferred as is the case with peptides in a bottom-up analysis. Another advantage is that alternative forms of a protein, e.g. post-translational modifications and splice variants, may be identified. However, top-down analysis has a disadvantage when compared to a bottom-up analysis in that many proteins can be difficult to isolate and purify. Thus, each protein in an incompletely separated mixture can yield, upon mass spectrometric analysis, multiple ion species, each species corresponding to a different respective degree of protonation and a different respective charge state, and each such ion species can give rise to multiple isotopic variants. Thus, methods are required for interpreting the resulting highly complex mass spectra.
Ion-ion reactions have found great utility in the field of biological mass spectrometry over the last decade, primarily with the use of electron transfer dissociation (ETD) to dissociate peptide/proteins and determine primary sequence information and characterize post-translational modifications.
Proton transfer, another type of ion-ion reaction, has also been used extensively in biological applications. Experimentally, proton transfer is accomplished by causing multiply-positively-charged protein ions (i.e., protein cations) from a sample to react with singly-charged reagent anions so as to reduce the charge state of an individual protein cation and the number of such charge states of the protein cations. These reactions proceed with pseudo-first order reaction kinetics when the reagent anions are present in large excess over the protein cation population. The rate of reaction is directly proportional to the square of charge of the protein cation (or other multiply-charged cation) multiplied by the charge on the reagent anion. The same relationship also holds for reactions of the opposite polarity, defined here as reaction between singly-charged reagent cations and a population of multiply-charged anions derived from a protein sample. This produces a series of pseudo-first order consecutive reaction curves as defined by the starting multiply-charged protein cation population. Although the reactions are highly exothermic (in excess of 100 kcal/mol), proton transfer is an even-electron process performed in the presence of 1 mtorr of background gas (i.e. helium) and thus does not fragment the starting multiply-charged protein cation population. The collision gas serves to remove the excess energy on the microsecond time scale (108 collisions per second), thus preventing fragmentation of the resulting product ion population.
Proton transfer reactions (PTR) have been used successfully to identify proteins in mixtures of proteins. This mixture simplification process has been employed to determine charge state and molecular weights of high mass proteins. PTR has also been utilized for simplifying product ion spectra derived from the collisional-activation of multiply-charged precursor protein ions. Although PTR reduces the overall signal derived from multiply-charged protein ions, this is more than offset by the significant gain in signal-to-noise ratio of the resulting PTR product ions. The PTR process is 100% efficient leading to only single series of reaction products, and no side reaction products that require special interpretation and data analysis.
Various aspects of the application of PTR to the analysis of peptides, polypeptides and proteins have been described in the following documents: U.S. Pat. No. 7,749,769 B2 in the names of inventors Hunt et al., U.S. Patent Pre-Grant Publication No. 2012/0156707 A1 in the names of inventors Hartmer et al., U.S. Pre-Grant Publication No. 2012/0205531 A1 in the name of inventor Zabrouskov; McLuckey et al., “Ion/Ion Proton-Transfer Kinetics: Implications for Analysis of Ions Derived from Electrospray of Protein Mixtures”, Anal. Chem. 1998, 70, 1198-1202; Stephenson et al., “Ion-ion Proton Transfer Reactions of Bio-ions Involving Noncovalent Interactions: Holomyoglobin”, J. Am. Soc. Mass Spectrom. 1998, 8, 637-644; Stephenson et al., “Ion/Ion Reactions in the Gas Phase: Proton Transfer Reactions Involving Multiply-Charged Proteins”, J. Am. Chem. Soc. 1996, 118, 7390-7397; McLuckey et al., “Ion/Molecule Reactions for Improved Effective Mass Resolution in Electrospray Mass Spectrometry”, Anal. Chem. 1995, 67, 2493-2497; Stephenson et al., “Ion/Ion Proton Transfer Reactions for Protein Mixture Analysis”, Anal. Chem. 1996, 68, 4026-4032; Stephenson et al., “Ion/Ion Reactions for Oligopeptide Mixture Analysis: Application to Mixtures Comprised of 0.5-100 kDa Components”, J. Am. Soc. Mass Spectrom. 1998, 9, 585-596; Stephenson et al., “Charge Manipulation for Improved Mass Determination of High-mass Species and Mixture Components by Electrospray Mass Spectrometry”, J. Mass Spectrom. 1998, 33, 664-672; Stephenson et al., “Simplification of Product Ion Spectra Derived from Multiply Charged Parent Ions via Ion/Ion Chemistry”, Anal. Chem., 1998, 70, 3533-3544 and Scalf et al., “Charge Reduction Electrospray Mass Spectrometry”, Anal. Chem. 2000, 72, 52-60. Various aspects of general ion/ion chemistry have been described in McLuckey et al., “Ion/Ion Chemistry of High-Mass Multiply Charged Ions”, Mass Spectrom. Rev. 1998, 17, 369-407 and U.S. Pat. No. 7,550,718 B2 in the names of inventors McLuckey et al. Apparatus for performing PTR and for reducing ion charge states in mass spectrometers have been described in U.S. Pre-Grant Publication No. 2011/0114835 A1 in the names of inventors Chen et al., U.S. Pre-Grant Publication No. 2011/0189788 A1 in the names of inventors Brown et al., U.S. Pat. No. 8,283,626 B2 in the names of inventors Brown et al. and U.S. Pat. No. 7,518,108 B2 in the names of inventors Frey et al. Adaptation of PTR charge reduction techniques to detection and identification of organisms has been described by McLuckey et al. (“Electrospray/Ion Trap Mass Spectrometry for the Detection and Identification of Organisms”, Proc. First Joint Services Workshop on Biological Mass Spectrometry, Baltimore, Md., 28-30 Jul. 1997, 127-132).
The product ions produced by the PTR process can be accumulated into one or into several charge states by the use of a technique known as “ion parking”. Ion parking uses supplementary AC voltages to consolidate the PTR product ions formed from the original variously protonated ions of any given protein molecule into a particular charge state or states at particular mass-to-charge (m/z) values during the reaction period. This technique can be used to concentrate the product ion signal into a single or limited number of charge states (and, consequently, into a single or a few respective m/z values) for higher sensitivity detection or further manipulation using collisional-activation, ETD, or other ion manipulation techniques. Various aspects of ion parking have been described in U.S. Pat. No. 7,064,317 B2 in the name of inventor McLuckey; U.S. Pat. No. 7,355,169 B2 in the name of inventor McLuckey; U.S. Pat. No. 8,334,503 B2 in the name of inventor McLuckey; U.S. Pat. No. 8,440,962 B2 in the name of inventor Le Blanc; and in the following documents: McLuckey et al., “Ion Parking during Ion/Ion Reactions in Electrodynamic Ion Traps”, Anal. Chem. 2002, 74, 336-346; Reid et al., “Gas-Phase Concentration, Purification, and Identification of Whole Proteins from Complex Mixtures”, J. Am. Chem. Soc. 2002, 124, 7353-7362; He et al., “Dissociation of Multiple Protein Ion Charge States Following a Single Gas-Phase Purification and Concentration Procedure”, Anal. Chem. 2002, 74, 4653-4661; Xia et al., “Mutual Storage Mode Ion/Ion Reactions in a Hybrid Linear Ion Trap”, J. Am. Soc. Mass. Spectrom. 2005, 16, 71-81; Chrisman et al., “Parallel Ion Parking: Improving Conversion of Parents to First-Generation Products in Electron Transfer Dissociation”, Anal. Chem. 2005, 77(10), 3411-3414 and Chrisman et al., “Parallel Ion Parking of Protein Mixtures”, Anal. Chem. 2006, 78, 310-316.